Process for stabilizing preparations of interfering viruses

ABSTRACT

A stabilized fluid preparation of interfering viruses containing a minor proportion of a mono- and/or disaccharide and a method of stabilization of interfering virus materials comprising mixing with the virus materials a minor proportion of a mono- and/or disaccharide and freeze-drying the resulting mixture.

O Unlted States Patent [151 3,674,864 .Angelucci July 4, 1972 [54}PROCESS FOR STABILIZING [56] References Cited PREPARATIONS F INTERFERINGUNITED STATES PATENTS VIRUSES 2,908,614 /1959 Muggleton et al. ..424/89X [72] Inventor; Romano Angelucci, Milan, Italy 3,186,908 6/1965Rightsel et a1. ..424/89 3,259,547 7/1966 Cole ...424/90 X Asslgnee=Carlo Erba i 6 Milan, Italy 3,485,718 12/1969 Baker ..195/1.5

[22] Filed: July 8, 1970 OTHER PUBLICATIONS 1 1 pp 53,325 Mizu TaniNature 198 (4875): 109- 110 A til 6, 1963 A P Simple Method forPurification of Influenza Virus. Related pp Data Reimer Science 152:1379- 1381 June 3, 1966 Influenza [63] Cmtinuatmmimpafl of Sen 687,135 1Virus Purification With the Zonal Ultracentrifuge."

abandoned Primary ExaminerShep K. Rose [30] Foreign Application PriorityData AtmmeyCuShman Darby & Cushman Dec. 16, 1966 Italy ..31189 A/66 [571ABSTRACT A stabilized fluid preparation of interfering virusescontaining [52] U.S.Cl ..424/90, 424/89, 1.5 a minor proportion of amonoand/0r disaccharide and a [51] Int. Cl. ..Cl2k l/08, C12k 1/00method of stabilization of interfering virus materials compris- [58]Field of Search ..424/89,90;195/1.5, 1.7 8 mixing with the virusmaterials a minor Proportion of a monoand/or disaccharide andfreeze-drying the resulting mixture.

8 Claims, No Drawings PROCESS FOR STABILIZING PREPARATIONS OFINTERFERING VIRUSFS CROSS-REFERENCE TO RELATED APPLICATION Thisapplication is a continuation-in-part of my copending application Ser.No. 687,135, filed Dec. 1, 1967 and now abandoned.

Interfering virus is a virus that has been modified so that if put incontact with susceptible cells, the interfering virus will enter thecell, will not multiply, but will prevent the multiplication of avirulent virus in that cell.

Interfering power is the power of such virus of preventing themultiplication, in a suitable medium, of any other virus also quitedifferent from the first one.

The present invention is, in general, directed to a process for thestabilization of the surface structures and interfering power ofpreparations of inactivated viruses, in particular influenza viruses,employable in the prophylaxis of viral diseases. Preparation of theinactivated viruses by treating viral suspensions with ultraviolet raysor heat is well documented in the prior art, e.g., to the method of W.Henle and K. Pauker (Virology 1958, 6, 181), U.S. Pat. No. 3,259,547,Australian Pat. No. 282,210. The irradiated or heat-treated virusesprepared according to the prior art method mentioned above are suspendedin buffer saline solutions of NaCl 0.9 percent by weight. The salinesuspended viruses are destroyed or damaged by subsequent freezing orlyophilization. For example, in tests carried out with suspensions ofinfluenza viruses APR 8; B Lee; FM 1, purified by adsorption andelusion'from barium sulphate followed by differential centrifugation, asignificant reduction in hemagglutinating titer after freezing andlyophilization was observed when the suspending medium was a salinesolution (NaCl, 0.9 percent) as shown in Table 1.

TABLE 1 I Variations of the hemagglutinating titer of influenza virusAPR 8, purified, irradiated, suspended in saline solutions and kept at25C.

Initial Hemagglutinating Titer 11 --Variations of the hemagglutinatingtiter of influenza virus APR 8, purified, irradiated and suspended insaline solution Initial hemagglutinating titer (BA 50%) l/l0240Hemagglutinating titer after lyophilization (HA titer 50%) 1/1280 Therehave been several suggested methods of stabilizing virus material, e.g.antigenic material, in the prior art. In U.S. Pat. No. 2,908,614, thereis disclosed the step of uniformly incorporating the sensitivebiological product in an aqueous dextran solution wherein the dextranhas a molecular range substantially within the range of from 10,000 to50,000. This reference also discloses that low molecular weight sugarssuch as glucose and lactose have the technical disadvantage that theyare singularly retentive of water thus making the freezedrying proceduremore hazardous to sensitive materials." The patentees found that byusing dextran alone it was particularly easy to overdry the sensitivematerials and thus kill or damage the organisms. It was found convenientto control the amount of residual moisture by incorporating, in thedextran medium, some inert substance which strongly retains water, e.g.glucose, which, in suitable concentration, enabled the optimum amount ofmoisture' to be retained (See U.S. Pat. No. 2,908,614, column 3, lines40-68). The patentees also reported that the stabilization of thevaccine could be obtained without the addition of the glucose byanalytical control of the drying step (see column 3, lines 69-72).

In U.S. Pat. No. 3,186,908, it was reported that when calciumlactobionate is added in minor proportion to virus antigen solutionsimproved stability of the virus was obtained. The patentees also suggestthe use of lactose in addition to the calcium lactobionate. The lactosefacilitates the drying of the antigen solution in a manner similar tothe glucose additive of U.S. Pat. No. 2,908,614.

It has now been found that an aqueous suspension of inactivated viruscan be stabilized from the detrimental harm incurred during freezing andlyophilization of the virus by incorporating a minor proportion of amonoand/or disaccharide into the aqueous suspension.

It is therefore an object of the invention to provide virus materialswhich have increased surface structures stability and increasedinterfering power especially during freeze-drying or when held instorage or in process for prolonged periods.

It is also an object to provide fluid virus materials which can besubjected to successive freezing and thawing without substantial loss ofinterfering potency.

A further object is to provide means for stabilizing virus materials.

These and other objects, features and advantages, which will be apparentfrom the following description of the invention, are realized byproviding stabilized interfering viruses obtained by purification of aviral suspension of virus obtainable by various known methods usingallantoic or amniotic liquids of embryonated eggs or liquids of diploidhuman cell cultured in vitro" infected with various viruses. Thepurified viruses are then inactivated and suspended in an aqueoussolution comprising a minor proportion of a monoand/or disaccharide. Theconcentration of inactivated virus in the aqueous solution is notcritical. The process of the present invention is applicable to bothsolutions containing small concentrations of virus and solutionscontaining large concentrations of virus.

Among the interfering viruses which have been found to be stabilized bythe addition of a minor proportion of monoand/or disaccharides to theaqueous suspension of viruses, there are influenza virus type A and typeB, parainfluenza virus type 3, New Castle disease virus, Sindbis virusand Vescicular stomatitis virus.

The monosaccharides which supply a stabilizing effect on the interferingviruses are glucose, mannose, laevulose, galactose, sorbose, talose,tagatose, gulose, mannitol and the like. The disaccharides which areeffective are gentiobiose, lactose, isomaltose, maltose, melibiose,nigerose, sucrose, sophorose, trehalose, xylobiose and the like.

The operative weight per cent ranges of the monoand/or disaccharides canvary from about 0.5 percent to about 25 percent, with the exception thatthose which have a solubility in water of less than 25 percent (forexample mannitol and lactose) can vary from about 0.5 percent up totheir maximum solubility in the solution. The preferred weight per centrange is of about 0.5 percent for the monosaccharides and of about 1percent for the disaccharides.

The invention is illustrated but not limited by the following examples.The concentration of ingredients is given in weight per volumepercentage, unless otherwise specified. In the examples, the suspensionof viruses were purified according to the method proposed by H.Mizutani, A Simple Method For Purification of Influenza Virus, Nature198, 4875, 109-1 10. The viral suspensions were first centrifuged at6,500 rpm. for 10 minutes and then the supernatant mixed with asuspension of 12.5 percent BaSO in bidistilled water. This mixture wasallowed to stand overnight at 4 C. and then slowly centrifuged. Thesediments were suspended into a 0.25 molar solution of sodium citratehaving a pH of 8.0. This mixture was then allowed to stand forapproximately 24 hours at 4 C. and centrifuged. The solution of sodiumcitrate containing the virus was recovered and dialyzed at 4 C. undercontinuous stirring against bidistilled water. The material so obtained,suitably diluted, was inactivated by irradiation with UN. rays and thencentrifuged at 43,500 rpm for minutes. The sediment was suspended intosolutions of various concentrations of monoand/or disaccharides andthese solutions were lyophilized.

EXAMPLE 1 Suspensions of influenza virus APR 8 purified as previouslysaid, were prepared in solutions of the following monoand disaccharides,then lyophilized. As controls equal suspensions were prepared in salinesolution (NaCl 0.9 by weight) and in buffer saline solution of Dulbeccoand Vogt. The results were as follows:

Hemagglutinating Hemagglutinating titer before titer afterlyophilization lyophilization Sucrose (1.0%) 1/5120 l/5120 Maltose(1.0%) 115120 1/5120 Lactose (1.0%) 1/5120 1/5120 Glucose (0.5%)

H 120 1/5/20 NaCl (0.9%) l/5l20 l/l280 Saline solution of H5120 l/l280Dulbecco and Vogt EXAMPLE 2 Influenza virus APR 8, which had beenpurified and irradiated with U.V. rays, were divided into two groups,the first suspended in a buffer saline solution of Dulbecco and Vogt andthe second suspended in a sucrose solution. These suspensions werelyophilized and redissolved by addition of a suitable volume ofdistilled water.

The stability of the two groups of inactivated virus solutions wasdetermined by first, inoculating embryonated eggs with doses of thesolutions, second, 16 hours after the first inoculation, inoculating theeggs with doses of active virus APR 8 and third, after periods of 24 and48 hours after incoulation with active virus, determining thehemagglutinating titer for a sample taken from the eggs. A set ofcontrol eggs was inoculated with the active virus only.

The hemagglutinating titer for samples taken from eggs which had beeninoculated by inactive virus suspended in 1 percent sucrose is shown inTable 2. The hemagglutinating titer for samples taken from eggs whichhad been inoculated by inactive virus suspended in the buffer salinesolutions of Dulbecco and Vogt is shown in Table 3.

The hemagglutinating titer for the control eggs which were inoculatedwith active virus only is shown in Table 4.

TA l3 LE 2 [interference induced from APR 8 virus inactivated with U.V.rays and suspended in 1% sucrose] ll 200 HA 50%. 1x10 EID 50% do lII 200HA 50%. 1X10 EID 50%.. d0

1 Negative.

TABLE 3 [Interference induced with APR 8 virus, inactivated with U.V.rays and prepared in bull'er saline solution of Dulbecco and Vogt] IIemagglutinating titers observed (lIA titers 50%) 24 hrs. alter 48 hrs.alter Inactive Active active virus active virus Al'lt H Allt xinoculation inoculation Group:

I :00 ll/\ 50% lxm" m I) 50% l/ltin [/320 II .500 [IA 50% lXllF' I'll l50",}. l/Ril l/MU Ill 'Jllll HA 130% IXltl I'll l) .iIlZ-j, l/t'vlll INet ntlve.

'lAliLlC 4 [Controls inoculated only with active A1 li 5 virus]llernaggltninathn: titers observed tllA titers 50 L 24 hrs. after Theabove data clearly shows the decreased interfering power of thepreparations of virus prepared in saline solutions in comparison withthe preparations in the sucrose solutions. The stabilizing efi'ect as topreparations of virus prepared in the sucrose solutions is dramatic.

What is claimed is:

1. A process for the stabilization against significant freezereductionof effective initial hemogglutination titers of inactivated virusmaterials which have been purified by adsorption and elution from bariumsulfate and concentrated by differential centrifugation and which areprophylactic against virus diseases, by interfering with themultiplication of virulent virus in susceptible cells, which comprisessuspending said virus materials in an aqueous solution consistingessentially of from 0.5 to 25 percent by weight of at least onesaccharide selected from the group consisting of glucose, mannose,laevulose, galactose, sorbose, talose, tagatose, gulose, mannitol,gentiobiose, lactose, isomaltose, maltose, melibiose nigerose, sucrose,sophorose, trehalose, and xylobiose, and subsequently freezing orfreeze-drying the suspended virus material without significant reductionin hemagglutination titer.

2. A process as in claim 1 wherein the aqueous solution of virusmaterials and saccharide is frozen.

3. A process as in claim 1 wherein the aqueous solutions of virusmaterials and saccharide is freeze-dried.

4. A process as in claim 3 wherein the virus materials is selected fromthe group consisting of influenza virus type A, influenza virus type B,parainfluenza virus type 3, New Castle disease virus, Sindbis virus andVescicular stomatitis virus.

5. A stabilized frozen or freeze-dried inactivated virus material inaccordance with claim 1 comprising said inactivated virus materialsuspended in an aqueous solution con sisting essentially of glucose,mannose, laevulose, galactose, sorbose, talose, tagatose, gulose,mannitol, gentiobiose, lactose, isomaltose, maltose, melibiose,nigerose, sucrose, sophorose, trehalose and xylobiose.

6. A stabilized interfering virus material as claimed in claim 5 whereinthe virus material is selected from the group consisting of influenzavirus type A, influenza virus type B, par-ainfluenza virus type 3, NewCastle disease virus, Sindbis virus and Vescicular stomatitis virus.

7. A stabilized interfering virus material produced by freeze-drying theproduct of claim 5.

8. A stabilized interfering virus material produced by freeze-drying theproduct of claim 6.

2. A process as in claim 1 wherein the aqueous solution of virusmaterials and saccharide is frozen.
 3. A process as in claim 1 whereinthe aqueous solutions of virus materials and saccharide is freeze-dried.4. A process as in claim 3 wherein the virus materials is selected fromthe group consisting of influenza virus type A, influenza virus type B,parainfluenza virus type 3, New Castle disease virus, Sindbis virus andVescicular stomatitis virus.
 5. A stabilized frozen or freeze-driedinactivated virus material in accordance with claim 1 comprising saidinactivated virus material suspended in an aqueous solution consistingessentially of glucose, mannose, laevulose, galactose, sorbose, talose,tagatose, gulose, mannitol, gentiobiose, lactose, isomaltose, maltose,melibiose, nigerose, sucrose, sophorose, trehalose and xylobiose.
 6. Astabilized interfering virus material as claimed in claim 5 wherein thevirus material is selected from the group consisting of influenza virustype A, influenza virus type B, parainfluenza virus type 3, New Castledisease virus, Sindbis virus and Vescicular stomatitis virus.
 7. Astabilized interfering virus material produced by freeze-drying theproduct of claim
 5. 8. A stabilized interfering virus material producedby freeze-drying the product of claim 6.